Encoded Microflakes

ABSTRACT

A digitally encoded microflake includes a polymer layer, which has a top surface and a bottom surface substantially parallel to the top surface. At least one of the top surface and the bottom surface is to be coupled to target-specific probes for bonding with a target analyte. The microflake is identified by a binary sequence of bits encoded by an edge outline on a plane substantially parallel to the top surface and the bottom surface. The bits in the binary sequence are encoded at respective predefined locations surrounding the edge outline.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/601,982 filed on Oct. 15, 2019, which claims the benefit ofU.S. Provisional Application No. 62/776,224 filed on Dec. 6, 2018, theentirety of which is incorporated by reference herein.

TECHNICAL FIELD

Embodiments of the invention relate to microflakes encoded with digitalcodes and the fabrication process of the microflakes.

BACKGROUND

With the development of precision medicine, personalized medicine, andpreventive medicine, there is a need in the field of in-vitro diagnosis(IVD) for performing assays on a large number of targets in biologicaltest samples. A multiplex approach, known as multiplex assays, enablesmultiple measurements to be simultaneously performed on multipledifferent targets to increase test throughput and reduce cost. Multiplexassays have a wide range of applications such as pathological diagnosis(e.g., infectious disease or oncology), food safety, human and animaldisease control, environmental monitoring, biomedical science research,drug screening and discovery, etc.

To conduct a multiplex assay, reagents such as probes are immobilized onthe solid-phase surface of microcarriers. Depending on the type of thedesired target, the probe can be an antibody, a protein, an antigen, aDNA, an RNA or another molecule with an affinity for the desired target.Microcarriers with different probes are added into a test sample, suchthat the probes and the corresponding targets in the test sample makespecific bonding. Excess substances not bounded to any probes may bewashed and removed. Detection antibodies or other suitable agents can beadded to the targets to form specific bonding. These detectionantibodies or suitable agents may contain a fluorophore, which emitslight upon excitation by a tight source. The emitted light allows thosetargets that are bounded to the probes to be observed and quantified.

A test sample in a multiplex assay typically contains multiple targetsof interest. To keep track of which targets are bounded to the probes onthe microcarriers, various approaches have been developed to encodemicrocarriers, such that microcarriers with different probes can bedistinguished from one another.

For example, one approach is to label different microcarriers withdifferent fluorescent dyes; however, the number of available fluorescentdyes is typically insufficient for multiplex assays where highthroughput is desired. Another approach is to encode a microcarrier witha pattern of opaque segments and transparent gaps, where the pattern isenclosed within the microcarrier.

Conventional approaches such as the ones described above generally haveseveral disadvantages such as high fabrication cost, insufficientreaction surface, poor performance, etc. Thus, there is a need forimproving the design and fabrication of encoded microcarriers.

SUMMARY

In one embodiment, a microflake is provided. The microflake includes apolymer layer, which has a top surface and a bottom surfacesubstantially parallel to the top surface. At least one of the topsurface and the bottom surface is to be coupled to target-specificprobes for bonding with a target analyte. The microflake is identifiedby a binary sequence of bits encoded by an edge outline on a planesubstantially parallel to the top surface and the bottom surface. Thebits in the binary sequence are encoded at respective predefinedlocations surrounding the edge outline.

In another embodiment, an apparatus for a multiplex assay is provided.The apparatus comprises a first microflake to form a target-specificbonding to a first target analyte, and a second microflake to form atarget-specific bonding to a second target analyte which is differentfrom the first target analyte. The first microflake is identified by afirst binary sequence, and the second microflake is identified by asecond binary sequence different from the first binary sequence. Thefirst binary sequence is encoded by a first edge outline on a firstplane substantially parallel to a top surface and a bottom surface ofthe first microflake, and bits in the first binary sequence are encodedat respective predefined locations surrounding the first edge outline.The second binary sequence is encoded by a second edge outline on asecond plane substantially parallel to a top surface and a bottomsurface of the second microflake, and bits in the second binary sequenceare encoded at respective predefined locations surrounding the secondedge outline.

In yet another embodiment, a semiconductor wafer is provided. Thesemiconductor wafer comprises a substrate; a plurality of firstmicroflakes on the substrate; and a plurality of second microflakes onthe substrate. Each first microflake is identified by a first binarysequence, and each second microflake is identified by a second binarysequence different from the first binary sequence. Each first microflakeis identified by a first binary sequence encoded by a first edge outlineon a plane substantially parallel to the substrate, and bits in thefirst binary sequence are encoded at respective predefined locationssurrounding the first edge outline. Each second microflake is identifiedby a second binary sequence encoded by a second edge outline on theplane, and bits in the second binary sequence are encoded at respectivepredefined locations surrounding the second edge outline.

Other aspects and features will become apparent to those ordinarilyskilled in the art upon review of the following description of specificembodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings in which likereferences indicate similar elements. It should be noted that differentreferences to “an” or “one” embodiment in this disclosure are notnecessarily to the same embodiment, and such references mean at leastone. Further, when a particular feature, structure, or characteristic isdescribed in connection with an embodiment, it is submitted that it iswithin the knowledge of one skilled in the art to effect such feature,structure, or characteristic in connection with other embodimentswhether or not explicitly described.

FIGS. 1A and 1B illustrate two opposite surfaces of a microflakeaccording to one embodiment.

FIGS. 2A and 2B illustrate two opposite surfaces of a microflakeaccording to another embodiment.

FIGS. 3A and 3B illustrate two opposite surfaces of a microflakeaccording to yet another embodiment.

FIGS. 4, 5, 6 and 7 illustrate a top view of encoded microflakesaccording to some embodiments.

FIGS. 8A, 8B, 8C and 8D illustrate a top view of microflakes encodedwith four different codes according to one embodiment.

FIGS. 9A, 9B and 9C illustrate microflakes, each of which uses adjacentangles to encode a group identifier according to some embodiments.

FIGS. 10A and 10B illustrate a three-dimensional view and across-section view, respectively, of a digitally encoded magneticmicroflake according to one embodiment.

FIGS. 10C and 10D illustrate a three-dimensional view and across-section view, respectively, of a digitally encoded magneticmicroflake according to another embodiment.

FIG. 10E illustrates an edge outline of a microflake according to oneembodiment.

FIGS. 11A, 11B, 11C and 11D illustrate steps of fabricating microflakesaccording to a first embodiment.

FIGS. 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H and 12I illustrate steps offabricating microflakes according to a second embodiment.

FIG. 13 is a schematic diagram illustrating a semiconductor waferincluding microflakes encoded with different codes according to oneembodiment.

FIG. 14 is a flow diagram illustrating a method for fabricatingdigitally encoded magnetic microflakes according to a first embodiment.

FIG. 15 is a flow diagram illustrating a method for fabricatingdigitally encoded magnetic microflakes according to a second embodiment.

FIG. 16 is a flow diagram illustrating a method for decoding digitallyencoded magnetic microflakes according to one embodiment.

FIG. 17 is a diagram illustrating a machine operable to execute asoftware product for decoding encoded microflakes according to oneembodiment.

DETAILED DESCRIPTION

In the following description, numerous specific details are set forth.However, it is understood that embodiments of the invention may bepracticed without these specific details. In other instances, well-knowncircuits, structures and techniques have not been shown in detail inorder not to obscure the understanding of this description. It will beappreciated, however, by one skilled in the art, that the invention maybe practiced without such specific details. Those of ordinary skill inthe art, with the included descriptions, will be able to implementappropriate functionality without undue experimentation.

Described herein are microflakes which may be used as carriers foranalyte detection and analysis in multiplex assays. Microflakes, alsoreferred to as digital magnetic flakes in some embodiments, can beidentified by their respective digital identifiers (also referred to asdigital code, or code). In one embodiment, the digital identifier of amicroflake is encoded by a sequence of notches and edge segments at theperiphery (i.e., edge) of the microflake. By detecting an edge outlineof a microflake, a detector can identify the code associated with themicroflake. More specifically, the edge outline, which outlines theperiphery of the microflake on a plane substantially parallel to the topsurface and the bottom surface of the microflake, encodes a binarysequence identifying the microflake.

When used in a multiplex assay, microflakes having the same code arecoupled to the same type of probe for capturing the same target analyte,and microflakes having different codes are coupled to different types ofprobes for capturing different target analytes.

In one embodiment, microflakes may include a magnetically responsivematerial to facilitate handling and collection. The magneticallyresponsive material may constitute substantially all of a microflake, aportion of a microflake, or only one component of a microflake. Theremainder of the microflake may include, among other things, polymericmaterial, coatings, and moieties which permit attachment of a probe.Examples of the magnetically responsive material include magnetic metalssuch as iron, nickel, cobalt, and alloys of rare-earth metals, but arenot limited thereto. In one embodiment, a magnetic metal strip may beembedded within a non-magnetic polymer layer of the microflake. Thenon-magnetic polymer layer may be substantially transparent.Alternatively, a microflake may be made of a magnetic photoresistmaterial; e.g., a mixture of magnetic particles and polymer. Themagnetic photoresist material may be substantially transparent. However,it is noted that the microflake may be made of materials different fromthe aforementioned materials.

A microflake may have any degrees of transparency. In one embodiment, amicroflake is substantially transparent. In another embodiment, amicroflake is at least partially transparent. As used herein, a materialis “substantially transparent” means that a high percentage of light canpass through the material (e.g., over 50% of light). A material is“partially transparent” when it is less transparent than a substantiallytransparent material and more transparent than an opaque material.

Microflakes may be fabricated using a wide variety of materials; forexample, resins and polymers. Examples of polymers include polystyrene,polydivnylbenzene, polymethylmethacrylate, poly-lactides,polyclycolides, polycaprolacton and copolymers thereof. Alternativematerials may also be used. In some embodiments described herein, thebody of a microflake is made of a substantially transparent polymermaterial. A microflake can be of any color.

In one embodiment, the digital codes of the microflakes described hereinare formed by a substantially transparent polymer material. When used ina multiplex assay, the microflakes that bond with fluorophore-labeledtarget analytes emit fluorescent light upon excitation by a light source(e.g., an ultraviolet light source). The light excitation may beperformed once and the resulting microflake images (which areilluminated by fluorescence) can be used for identifying theirrespective codes as well as for the analysis (e.g., quantification) ofthe target analytes. The edge outlines of these fluorescence-illuminatedmicroflake images can be easily detected against a dark background.Those microflakes that do not emit fluorescent light upon excitation donot bond with any target analytes, and, therefore, do not need to bedecoded.

By contrast, a conventional microcarrier encoded with an embedded opaquematerial may need two steps of light excitations. In the first step, allof the microcarriers in a sample are illuminated by a first light sourcein a first light spectrum (e.g., ultraviolet light) for the purpose ofanalyzing (e.g., quantifying) the target analytes. In the second step,all of the microcarriers in the sample are again illuminated by a secondlight source in a second light spectrum (e.g., visible light) for thepurpose of identifying their codes. In the first step thosemicrocarriers bonded with target analytes are fluorescence-illuminatedagainst a dark background; however, the code patterns formed by theembedded opaque material typically cannot be decoded under thefluorescence. One reason is that the fluorescence is emitted from themicrocarrier surface, which does not provide sufficient illumination fordistinguishing embedded opaque code patterns. Therefore, the second stepis generally needed by the conventional designs to illuminate themicrocarriers in visible light. The additional step (i.e., the secondstep) increases the complexity of the decoding process.

Furthermore, in a multiplex assay where a microflake has probes on boththe top and bottom surfaces, all of these probes can capture afluorophore-labeled target analyte and the microflake can be illuminatedon both surfaces by the fluorescence. Higher transparency of themicroflake means that not only the fluorescence from the top (front)surface can be detected, but also the fluorescence from the bottom(back) surface can pass through the microflake body and be detected.Thus, a detector may read the code identifying the microflake moreeasily from the image of the fluorescence-illuminated microflake.However, it is understood that the microflake encoding schemes describedherein are applicable to microflakes of any degree of transparency.

A microflake is a substantially flat microcarrier with substantiallyplanar top and bottom surfaces. The size of a microflake, when measuredfrom the longest dimension (e.g., the diameter or the diagonal length atthe top or bottom surface) is typically in the range of a few μm tohundreds μm. The thickness of a microflake is typically in the range of1 μm to 10 μm.

In one embodiment, a microflake is encoded by one or more types ofnotches at its edge. A first-type notch is an orientation indicator thatidentifies an orientation of the microflake; e.g., the top surface orthe bottom surface. The first-type notch further indicates a startingpoint of a binary sequence and a direction along which the binarysequence is to be read, where the binary sequence is the codeidentifying the microflake. A second-type notch is used for encoding themicroflake. In one embodiment, a microflake is encoded by its edgeoutline which includes a sequence of edge segments and second-typenotches. The sequence of edge segments and second-type notches encodesthe binary sequence of zeros and ones.

The first-type and the second-type notches may have V-shape, U-shape,square-shape, rectangular shape or the like, at the edge of themicroflake. In some embodiments, a first-type notch may be formed byremoving a corner of a polygon. Each of the notches (including thefirst-type notches and the second-type notches) extends from the topsurface to the bottom surface of the microflake.

In the following description, the term “substantially parallel” is usedherein to mean that two lines, layers or planes are parallel or may bedeviated slightly from being parallel. The slight deviation may comefrom the fabrication process and is within an allowable tolerance range.Thus, the terms “parallel” and “substantially parallel” areinterchangeable in this disclosure to mean that two or more lines,layers, and/or planes are parallel within an allowable tolerance range.Similarly, the terms “substantially planar,” “substantially vertical”and “substantially perpendicular” are used to indicate “planar,”“vertical” and ‘perpendicular,” respectively, within an allowabletolerance range.

FIGS. 1A, 1B, 2A, 2B, 3A and 3B are schematic diagrams illustratingmicroflakes 100, 200 and 300 according to some embodiments. Each of themicroflakes 100, 200 and 300 has only the first-type notch (11, 21, and31, respectively) present at the edge. FIGS. 1A, 2A and 3A illustratethe top surfaces of the microflakes 100, 200 and 300, respectively, andFIGS. 1B, 2B and 3B illustrate the bottom surfaces of the microflakes100, 200 and 300, respectively.

It is noted that the terms “top” and “bottom” are used herein to namethe two opposite surfaces of a microflake. In use (e.g., during amultiplex assay process), the bottom surface may be on top while the topsurface may be on the bottom of a microflake, or vice versa.

The examples herein illustrate that a microflake can be of any shape asits base shape; e.g., a polygon, a circle, or the like. It is noted thatthe base shape of a microflake refers to the shape of a microflakewithout any of the notches.

The first-type notches 11, 21, and 31 define the orientation (e.g.,front or back, top or bottom) of the respective microflakes 100, 200 and300. A detector can identify whether an observed surface of a microflakeis the top surface or the bottom surface of the microflake; e.g., byidentifying the lengths of the two adjacent sides or the two adjacentangles of the first-type notch. After the detector identifies whichsurface of the microflake is being or has been observed, the detector oranother device can then determine the direction in which the code at theedge of the microflake is to be read.

FIG. 1A further shows that the first-type notch 11 may be formed byremoving a triangular portion of a corner (shown in dotted lines). Thepart of the edge outline that defines the first-type notch 11 may be astraight line (which is shown in FIG. 1A), a curved line, or anothershaped line. As shown in FIG. 1A and FIG. 1B, the first-type notch 11forms an asymmetric relationship with its two adjacent sides (e.g., “a”and “d”), where a and d have different lengths (e.g., a<d). Thisdifference in adjacent side lengths helps a detector to determinewhether an observed surface of a microflake is the top surface or thebottom surface of the microflake. For instance, an identifier may beencoded in the form of a sequence of second-type notches and/or edgesegments of a microflake. The sequence read clockwise from the topsurface of the microflake can be different from the sequence readclockwise from the bottom surface of the microflake. To correctly readand interpret a detected sequence, the detector first uses thefirst-type notch to identify the surface of the microflake. In theexample of FIGS. 1A and 1B, the detector may identify whether theobserved surface is the top surface or bottom surface by reading theside lengths clockwise from the first-type notch. That is, if thelengths are read as a→b→c→d, then the observed surface is the topsurface. However, if the lengths are read as d→c→b→a, then the observedsurface is the bottom surface. In one embodiment, the detector maycompare the lengths of the first-type notch's two adjacent sides byfollowing a predetermined direction (e.g., clockwise) to determine whichsurface of the microflake is being or has been observed. When read inthe clockwise direction, if the first-encountered adjacent side (e.g.,a) is shorter than the second-encountered adjacent side (e.g., d), thenaccording to the example of FIG. 1A, the observed surface is the topsurface. Similarly, when read in the clockwise direction, if thefirst-encountered adjacent side (e.g., d) is longer than thesecond-encountered adjacent side (e.g., a), then according to theexample of FIG. 1B, the observed surface is the bottom surface.

Alternatively or additionally, the first-type notch 11 may form anasymmetric relationship with its two adjacent angles (e.g., θ₁ and θ₂),where θ₁ and θ₂ have different values (e.g., θ₁>θ₂). Each adjacent angleis defined by a side of the first-type notch 11 and one adjacent side ofthe microflake 100. This difference in the adjacent angles helps adetector to determine whether an observed surface of a microflake is thetop surface or the bottom surface of the microflake. For instance, thedetector may determine the observed surface to be the top surface if thefirst-encountered angle read clockwise from the first-type notch 11 isOi, or if the two adjacent angles read clockwise from the first-typenotch 11 is θ₁→θ₂, or if the two adjacent angles read clockwise from thefirst-type notch 11 is a larger angle (e.g., θ₁) followed by a smallerangle (e.g., θ₂). Similar determinations may be made regarding thebottom surface of the microflake 100.

FIG. 2A shows that the first-type notch 21 may be formed by removing anon-isosceles triangular portion from a corner of the microflake 200(shown in dotted lines). As shown in FIG. 2A and FIG. 2B, the first-typenotch 21 forms an asymmetric relationship with its two adjacent sides(e.g., “a” and “d”), where a and d have different lengths (e.g., a>d).Alternatively or additionally, the first-type notch 21 may form anasymmetric relationship with its two adjacent angles (e.g., θ₁ and θ₂),where θ₁ and θ₂ have different values (e.g., θ₁<θ₂). This difference inadjacent side lengths and/or adjacent angles helps a detector todetermine whether an observed surface of a microflake is the top surfaceor the bottom surface of the microflake 200.

FIG. 3A shows that the first-type notch 31 may be formed by removing awedge portion from the microflake 300 (shown in dotted lines). As shownin FIG. 3A and FIG. 3B, the first-type notch 31 forms an asymmetricrelationship with its two adjacent angles (e.g., θ₁ and θ₂), where θ₁and θ₂ have different values (e.g., θ₁<θ₂). The first-type notch 31 mayalso form an asymmetric relationship with the two sides of the wedgeportion that forms the first-type notch 31. This difference in adjacentangles and/or side lengths helps a detector to determine whether anobserved surface of a microflake is the top surface or the bottomsurface of the microflake 300.

FIGS. 4-7 illustrate a top view of encoded microflakes according to someembodiments. The code of a microflake is a binary sequence of ones andzeros, represented by the presence or absence of second-type notches atthe edge of the microflake. To simplify the description, the followingexamples assume that the presence of a second-type notch indicates abinary “1” value and the absence of a second-type notch indicates abinary “0” value. An absent second-type notch is equivalent to an edgesegment that is present at the same location. It is understood that therepresentation of “1” and “0” may be reversed in alternativeembodiments. The second-type notches can be of any shape, as long as itis different from the shape of the first-type notch.

In one embodiment, a detector may read the code of a microflake asfollows. Starting from the first-type notch, the detector may check theedge outline of the microflake at a fixed interval of physical distance(e.g., a unit length X) to identify whether a second-type notch ispresent or absent. The detector may start reading the code at apredetermined distance from one end of the first-type notch, in apredetermined direction (e.g., clockwise or counter-clockwise) based onwhether the observed surface is the top surface or the bottom surface.For example, the code (from the least significant bit (LSB) to the mostsignificant bit (MSB)) may be defined to be the bit sequence readclockwise from the top surface of the microflake. An alternativedefinition may also be used.

FIG. 4 illustrates a top view of an encoded microflake 400 according toone embodiment. In this example, the code from the LSB to the MSB isread clockwise in this top view from the first-type notch 41 in thedirection of dotted arrow 45. The shape of each second-type notch inthis example is defined by its two substantially parallel sides (onlyone of the second-type notches is labeled 42 to simplify theillustration) and a bottom side adjoining the two substantially parallelsides. The portion of the edge outline, where neither a first-type notchnor a second-type notch is present, is referred to as an edge segment(only one of the edge segments is labeled 43 to simplify theillustration). The top view of the microflake 400 shows an edge outline,which is a combination of edge segments, sides of the first-type notchand the second-type notches. In this example, each edge segment oflength X is interpreted as a “0” bit and each second-type notch oflength X is interpreted as a “1” bit. The length of a second-type notchmay be defined as the length measured across the two sides of itsopening indicated by “w” (only one is labeled to simplify theillustration). Two consecutive second-type notches may occupy a corner;e.g., the lower-left corner as shown in the top view. The edge outlineof the microflake 400, represented by a sequence of lengths, in theorder of being read (clockwise) is as follows:3X→(2X)→X→6X→(2X)→4X→(2X)→(2X)→X→(2X)→2X→(2X)→2X→4X→(2X)→2X→(2 X)—>X,where the value in the parenthesis indicates the length of acorresponding second-type notch (i.e., representing one or more “1” s).Accordingly, the above sequence of lengths can be interpreted into thedigital code (from LSB to MSB) identifying the microflake 400 as:011001100000011001101111000011000000011000, which is read from right toleft to correspond to the aforementioned clockwise order, and where LSBis the rightmost bit and MSB is the leftmost bit.

FIG. 5 illustrates a top view of an encoded microflake 500 according toanother embodiment. In this example, the code from the LSB to the MSB isread clockwise in this top view from the first-type notch 51 in thedirection of dotted arrow 55. Each second-type notch in this example isdefined by two sides that join at one end (only one of the second-typenotches is labeled 52 to simplify the illustration). From the top view,each second-type notch has a triangular shape. The edge outline of themicroflake 500 can be read and interpreted into a digital code similarlyto that of the microflake 400 in FIG. 4.

FIG. 6 and FIG. 7 illustrate a top view of an encoded microflake 600 andan encoded microflake 700, respectively, according to some otherembodiments. The microflakes 600 and 700 have first-type notches 61 and71, respectively. The codes associated with the microflakes 600 and 700can be read and interpreted into respective digital codes according tothe aforementioned description in connection with the microflakes 400and 500.

The second-type notches shown in FIGS. 4-7 have symmetrical shapes. Inalternative embodiments, a second-type notch may have an asymmetricalshape; e.g., one side is longer than the other side. A second-type notchcan have any shape, as long as the shape is distinguishable from theshape of a first-type notch.

In one embodiment, the opening of a second-type notch can be of anylength (measured across the two sides of its opening) that is an integermultiple of a predetermined unit length (e.g., denoted by X in FIGS.4-7). In some embodiments, the length of a second-type notch's openingdefines the number of consecutive bits having a first binary value inthe binary sequence (i.e., the digital code), and the length of eachedge segment defines the number of consecutive bits having a secondbinary value in the binary sequence. In some embodiments, the length ofa second-type notch is limited to a predetermined length; e.g., one ortwo unit lengths. For example, a microflake may include a sequence ofconsecutive unit-length second-type notches to represent a long sequenceof “1s”.

FIGS. 8A and 8B illustrate a top view of two encoded microflakes 810 and820, respectively, according to some embodiments. Each of themicroflakes 810 and 820 has a first-type notch at the upper-right cornerin the top view. The microflake 810 is encoded with a sequence of onesusing a sequence of consecutive unit-length second-type notches. In theexample of FIG. 8A, the second-type notches are formed withinpredetermined portions of the edge outline (e.g., P1, P2, P3 and P4),avoiding the vicinity of the corner areas. One side of the second-typenotch (representing the MSB or the LSB of the code) may adjoin a side ofthe first-type notch. The microflake 820, which is without anysecond-type notches, is encoded with a sequence of zeros.

FIGS. 8C and 8D illustrate the top view of two encoded microflakes 830and 840, respectively, according to some embodiments. Each of themicroflakes 830 and 840 has a first-type notch at the upper-right cornerin the top view. Each of the microflakes 830 and 840 is encoded with asequence of alternating ones and zeros using a sequence of second-typenotches and edge segments. In these examples, the codes (from the LSB tothe MSB) associated with the microflakes 830 and 840, as shown in FIGS.8C and 8D, are read and interpreted clockwise from the respectivefirst-type notches.

FIGS. 8C and 8D further illustrate predefined locations surrounding theedge outline of the microflakes 830 and 840. The predefined locations ofbits 0-29 of the code are marked on the dotted lines. In theseembodiments, a bit has a first value if its predefined location has nosecond-type notch, and a bit has a second value if its predefinedlocation has a second-type notch present. The spacing between adjacentpredefined locations may be any value, as a detector does not measurethe spacing in determining the bit values of a code. When reading thecode, a detector may be provided with a detection template with markingof these predefined locations. The detection template may be overlaid onthe image of a microflake's edge outline for the detector to determinewhether a second-type notch is present at these locations. A detectiontemplate may have the same shape as the base shape of the microflake,with location markings for the bits. Examples of the detection templatesmay be the dotted rectangles 880 and 890 with the location markings inFIGS. 8C and 8D. A detection template may be larger, smaller or of thesame size as the base shape of the microflake, as long as its locationmarkings indicate the bit locations.

In one embodiment, a microflake may be identified by a group identifierin addition to a binary sequence formed by its edge segments andsecond-type notches. The group identifier may be encoded by acombination of the adjacent angles of the first-type notch, such as θ₁and θ₂ shown in FIGS. 8A-8D. For ease of illustration, FIGS. 9A, 9B and9C illustrate examples of three groups without any second-type notches.It is understood that the group identifiers can be combined with any ofthe binary sequences described herein to form a digital code of amicroflake.

FIG. 9A illustrates a first example of a group identifier in which thetwo adjacent angles have the same value (e.g., θ₁=θ₂=135°). This isapplicable to a microflake which has asymmetrical sides (e.g., arectangle). FIG. 9B illustrates a second example of a group identifier,e.g., θ₁=150°, θ₂=120°. FIG. 9C illustrates a third example of a groupidentifier, e.g., θ₁=120°, θ₂=150°. In one embodiment, different valuesof angle_diff=(θ₁-θ₂) correspond to different group identifiers. In oneembodiment, the value of angle_diff may be represented by a number ofbits, where the number is determined according to the number of groups.

FIGS. 10A and 10B illustrate a three-dimensional (3D) view and across-sectional view of a microflake 1000, respectively, according toone embodiment. The cross-sectional view of the microflake 1000 in FIG.10B shows a top surface 1010, a bottom surface 1020 substantiallyparallel to the top surface 1010, and an edge 1030 (i.e., the periphery)surrounding the top surface 1010 and the bottom surface 1020. The topsurface 1010 and the bottom surface 1020 are substantially planar; e.g.,substantially parallel to the horizontal plane spanned by the X-Y axes(i.e., the X-Y plane) as shown. The edge 1030 is substantiallyperpendicular to the two surfaces 1010 and 1020; e.g., substantiallyparallel to the Z-axis as shown. As shown in FIG. 10B, the edge 1030extends vertically between the top surface 1010 and the bottom surface1020. In one embodiment, the top surface 1010 and the bottom surface1020 may be symmetric with respect to the X-Y plane. When used in amultiplex assay, either one or both of the surfaces 1010 and 1020 may becoupled to reagents (e.g., probes) for bonding with target analytes.

In one embodiment, the microflake 1000 is made of a magnetic polymermaterial, which is substantially transparent. The magnetic polymermaterial may be produced by mixing magnetic metal particles into thepolymer. The microflake 1000, which is magnetically responsive, can beeasily manipulated and handled with magnetic fields during the processof a multiplex assay.

FIG. 10C illustrates an edge outline 1080 of the microflake 1000according to one embodiment. The edge outline 1080 encodes themicroflake 1000 according to any one of the aforementioned edge encodingschemes. FIG. 10C shows that the edge outline 1080 outlines theperiphery of both the top surface 1010 and the bottom surface 1020 on aplane (e.g., the X-Y plane in this example). The plane is substantiallyparallel to the top surface 1010 and the bottom surface 1020. Thus, adetector can detect the same code of the microflake 1000 from bothsurfaces 1010 and 1020. The same binary sequence of the code may be readclockwise from one of the surfaces 1010 and 1020, and counter-clockwisefrom the other one of the surfaces 1010 and 1020.

FIGS. 10C and 10D illustrate a 3D view and a cross-sectional view of amicroflake 1060, respectively, according to another embodiment. Similarto the microflake 1000 in FIGS. 10A and 10B, the microflake 1060 alsoincludes a top surface 1016, a bottom surface 1026 substantiallyparallel to the top surface 1016, and an edge 1036 surrounding the topsurface 1016 and the bottom surface 1026. In addition, the microflake1060 has a magnetic metal strip 1050, e.g., nickel, cobalt, iron, or thelike, embedded therein. The magnetic metal strip 1050 is anon-transparent (i.e., opaque) layer embedded within the microflake1060. The magnetic metal strip 1050 may be sandwiched between a toptransparent polymer layer and a bottom transparent polymer layer of themicroflake 1060. In one embodiment, the magnetic metal strip 1050 islocated at a geometric center of the microflake 1060. The geometriccenter location helps to reduce the torque on the microflake 1060 whenthe microflake 1060 is picked up or collected with a magnetic fieldduring a multiplex assay process. Similar to the microflake 1000 in FIG.10A, the microflake 1060 is encoded by its edge outline which outlinesthe periphery of both the top surface 1016 and the bottom surface 1026on a plane substantially parallel to the top surface 1016 and the bottomsurface 1026.

FIG. 10E illustrates an edge outline 1080 of the microflake 1000 or 1060according to one embodiment. The edge outline 1080 encodes themicroflake 1000 according to any one of the aforementioned edge encodingschemes. Using the microflake 1000 as an example, FIG. 10E shows thatthe edge outline 1080 outlines the periphery (i.e., edge) of both thetop surface 1010 and the bottom surface 1020 on a plane (e.g., the X-Yplane in this example). The plane is substantially parallel to the topsurface 1010 and the bottom surface 1020. Thus, a detector can detectthe same code of the microflake 1000 from either one of the surfaces1010 and 1020. The same binary sequence of the code may be readclockwise from one of the surfaces 1010 and 1020, and counter-clockwisefrom the other one of the surfaces 1010 and 1020.

The aforementioned microflakes may be fabricated based on techniquesdeveloped for semiconductor fabrication and/or micro-electro-mechanicalsystems (MEMS) fabrication. For example, the microflake fabricationdescribed below uses substrate materials, sacrificial materials, andphotoresist materials that are known in the art of the semiconductorand/or MEMS fabrication. A number of steps known to skilled persons inthe field of semiconductor and/or MEMS fabrication are omitted below tosimplify the illustration. For example, omitted steps may includesurface cleaning to prepare surfaces for subsequent processing,soft-baking to remove solvents, etc. The fabrication processes describedbelow are simpler, cost less and reserve more reaction areas comparedwith a process for fabricating conventional microcarriers.

FIGS. 11A, 11B, 11C and 11D illustrate steps of fabricating microflakesaccording to a first embodiment. FIG. 14 is a flow diagram illustratinga method 1400 for fabricating microflakes according to the firstembodiment. Examples of the microflakes fabricated according to thefirst embodiment include, but are not limited to, the microflake 1000 ofFIGS. 10A and 10B. Referring to FIGS. 11A-11D and 14, the method 1400starts at step 1410 with depositing a sacrificial layer 1120 on top of asubstantially planar substrate 1110 on a wafer. Subsequently at step1420, a magnetic photoresist layer 1130 (e.g., a magnetic polymer layer)is deposited on top of the sacrificial layer 1120. The magneticphotoresist layer 1130 may be substantially transparent or at leastpartially transparent in some embodiments. A mask layer 1140 whichcontains microflake patterns is used, at step 1430, to pattern themagnetic photoresist layer 1130 into microflakes. The mask layer 1140selectively exposes areas of the magnetic photoresist layer 1130 toultraviolet (UV) light to thereby define the individual microflakes1100. Each microflake 1100 is encoded by its edge outline which outlinesa 2D periphery of both the top surface and the bottom surface of themicroflake 1100 on a plane that is substantially parallel to the top andbottom surfaces. In one embodiment, the mask layer 1140 may definemicroflakes encoded with different codes on the wafer. Thus, microflakeswith different codes may be fabricated at the same time on the samewafer. At step 1440, the sacrificial layer 1120 is etched or otherwiseremoved to release the microflakes 1100 from the substrate 1110.

FIGS. 12A-12I illustrate steps of fabricating microflakes according to asecond embodiment. FIG. 15 is a flow diagram illustrating a method 1500for fabricating microflakes according to the second embodiment. Examplesof the microflakes fabricated according to the second embodimentinclude, but are not limited to, the microflake 1060 of FIGS. 10C and10D. Referring to FIGS. 12A, 12B and 15, the method 1500 starts at step1510 with depositing a sacrificial layer 1220 on top of a substantiallyplanar substrate 1210 on a wafer. Subsequently at step 1520, aphotoresist layer 1230 a (e.g., a polymer layer) is deposited on top ofthe sacrificial layer 1220. The photoresist layer 1230 a may besubstantially transparent or at least partially transparent in someembodiments. A mask layer 1240 which contains microflake patterns isused, at step 1530, to pattern the photoresist layer 1230 a intomultiple blocks 1230, where each block 1230 is to be part of acorresponding microflake. The mask layer 1240 selectively exposes areasof the photoresist layer 1230 to UV light to thereby define theindividual blocks 1230.

A lift-off process may be performed to deposit a magnetic metal layer oneach bottom portion. FIGS. 12C-12F illustrate the lift-off processaccording to one embodiment. A second sacrificial material 1250 isblanket-deposited on top of the remaining structure. The secondsacrificial material 1250 is patterned to expose at least a portion ofthe top surface of each block 1230. A thin layer of a magnetic metal1260 is deposited to cover the entire top of the patterned sacrificialmaterial 1250 and the exposed blocks 1230, by Physical Vapor Deposition(PVD), Chemical Vapor Deposition (CVD), etc. The second sacrificialmaterial 1250 is then stripped off, e.g., by using a chemical solutionor alternative means. Thus, at step 1540, a strip of the magnetic metallayer 1260 is formed on at least a portion of the top surface of eachblock 1230.

Referring to FIGS. 12G-12I and 15, at step 1550, another photoresistlayer 1270 a (e.g., a polymer layer) is blanket-deposited over theremaining structure; i.e., over the blocks 1230 and over the magneticmetal layer 1260 on top of each block 1230. In one embodiment, thephotoresist layer 1270 a may be the same material as the photoresistlayer 1230 a. The photoresist layer 1270 a may be substantiallytransparent or at least partially transparent in some embodiments. Atstep 1560, a mask layer 1280 which contains microflake patterns is usedto pattern the photoresist layer 1270 a into microflakes 1200. Eachmicroflake 1200 is encoded by its edge outline which outlines a 2Dperiphery of both the top surface and the bottom surface of themicroflake 1200 on a plane that is substantially parallel to the top andbottom surfaces. In one embodiment, the mask layer 1280 may definemicroflakes encoded with different codes on the wafer. Thus, microflakeswith different codes may be fabricated at the same time on the samewafer. At step 1570, the sacrificial layer 1220 is etched or otherwiseremoved to release the microflakes 1200 from the substrate 1210.

FIG. 13 is a schematic diagram illustrating a semiconductor wafer 1300having a plurality of microflakes on a substrate 1350 according to oneembodiment. The example of FIG. 13 shows that microflakes with differentcodes can be fabricated on the same wafer; e.g., by patterning themicroflakes with a mask layer that defines different edge outlinescorresponding to the different codes. Non-limiting examples of thepatterning step have been provided at step 1430 in FIG. 14 and step 1560in FIG. 15. For example, the wafer 1300 may be fabricated to include themicroflakes 810, 820, 830 and 840 (FIGS. 8A-8D). In one embodiment, thewafer 1300 may include multiple partitions, with each partitionincluding a group of microflakes of the same code. It is understood thatin alternative embodiments, a wafer may include microflakes having moredifferent codes, or fewer different codes, than what is shown in FIG.13.

The aforementioned microflakes may have a number of uses, including butnot limited to, a multiplex assay kit. The multiplex assay kit includesat least a first microflake according to any of the above embodiments toform a target-specific bonding to a first target analyte, and a secondmicroflake according to any of the above embodiments to form atarget-specific bonding to a second target analyte which is differentfrom the first target analyte. The first microflake is identified by afirst binary sequence, and the second microflake is identified by asecond binary sequence. The first binary sequence is encoded by a firstedge outline, which outlines a periphery of the first microflake on afirst plane substantially parallel to a top surface and a bottom surfaceof the first microflake. The second binary sequence is encoded by asecond edge outline, which outlines a periphery of the second microflakeon a second plane substantially parallel to a top surface and a bottomsurface of the second microflake.

FIG. 16 is a flow diagram illustrating a method 1600 for decodingdigitally encoded magnetic microflakes according to one embodiment. Themethod 1600 may be performed by a detector, such as a machine to bedescribed in connection with FIG. 17. The method 1600 begins at step1610 where the detector obtains an image containing a plurality ofilluminated microflakes. At step 1620, the detector detects an edgeoutline of each illuminated microflake. From the edge outline, thedetector at step 1630 identifies a first-type notch to determine anorientation of a corresponding microflake. At step 1630, the detectordetects the presence (or absence) of second-type notches to decode theedge outline into a binary sequence. The binary sequence is the digitalcode identifying the corresponding microflake.

FIG. 17 is a diagram illustrating a machine 1700 operable to execute asoftware product 1780 for decoding the encoded microflakes according toone embodiment. The software product 1780 may be stored in amachine-readable medium (such as the non-transitory machine-readablestorage media 1720, also referred to as a computer-readable medium, aprocessor-readable medium, or a computer-usable medium having acomputer-readable program code embodied therein). The non-transitorymachine-readable medium 1720 may be any suitable tangible mediumincluding a magnetic, optical, or electrical storage medium including adiskette, compact disk read-only memory (CD-ROM), digital versatile discread-only memory (DVD-ROM) memory device (volatile or non-volatile) suchas hard drive or solid-state drive, or another storage mechanism. Themachine-readable medium 1720 may contain various sets of instructions,code sequences, configuration information, or other data, which, whenexecuted, cause a processor 1710 to decode the encoded microflakes in animage 1750; e.g., according to the method 16 of FIG. 16. The image 1750may be obtained by an I/O device 1730 of the machine 1700. From theimage 1750, the machine 1710 may generate an output that includes atleast the identified codes associated with the microflakes. Those ofordinary skill in the art will appreciate that other instructions andoperations necessary to implement the described embodiments may also bestored on the machine-readable medium 1720. Software running from themachine-readable medium may interface with circuitry to perform thedescribed tasks.

While the invention has been described in terms of several embodiments,those skilled in the art will recognize that the invention is notlimited to the embodiments described, and can be practiced withmodification and alteration within the spirit and scope of the appendedclaims. The description is thus to be regarded as illustrative insteadof limiting.

1. An apparatus for a multiplex assay, comprising: a first microflake toform a target-specific bonding to a first target analyte; and a secondmicroflake to form a target-specific bonding to a second target analytewhich is different from the first target analyte, wherein the firstmicroflake is identified by a first binary sequence, and the secondmicroflake is identified by a second binary sequence different from thefirst binary sequence, and the first binary sequence is encoded by afirst edge outline on a first plane substantially parallel to a topsurface and a bottom surface of the first microflake, and bits in thefirst binary sequence are encoded at respective predefined locationssurrounding the first edge outline, the second binary sequence isencoded by a second edge outline on a second plane substantiallyparallel to a top surface and a bottom surface of the second microflake,and bits in the second binary sequence are encoded at respectivepredefined locations surrounding the second edge outline, each of thefirst edge outline and the second edge outline includes a first-typenotch which indicates a starting point and a direction along which thefirst binary sequence or the second binary sequence is to be read, andeach of the first microflake and the second microflake is furtheridentified by a group identifier which is encoded by adjacent angles ofthe first-type notch, and wherein different combinations of the adjacentangles encode different group identifiers.
 2. The apparatus of claim 1,wherein each of the first microflake and the second microflake includesa substantially transparent polymer layer.
 3. The apparatus of claim 2,wherein the substantially transparent polymer layer is a magneticpolymer layer.
 4. The apparatus of claim 2, wherein a magnetic metalstrip is embedded within and located at a geometric center of thesubstantially transparent polymer layer.
 5. The apparatus of claim 1,wherein each of the first edge outline and the second edge outlineincludes the first-type notch as an orientation indicator, and at leastone of the first edge outline and the second edge outline includes asecond-type notch representing a binary value in the first binarysequence or the second binary sequence.
 6. The apparatus of claim 1,wherein presence of a second-type notch at a predefined location of eachof the first edge outline and the second edge outline corresponds to afirst binary value, and absence of the second-type notch at thepredefined location of each of the first edge outline and the secondedge outline corresponds to a second binary value.
 7. An apparatus of asemiconductor wafer, comprising: a substrate; a plurality of firstmicroflakes on the substrate, each first microflake identified by afirst binary sequence; and a plurality of second microflakes on thesubstrate, each second microflake identified by a second binary sequencedifferent from the first binary sequence, wherein each first microflakeis identified by a first binary sequence encoded by a first edge outlineon a plane substantially parallel to the substrate, and bits in thefirst binary sequence are encoded at respective predefined locationssurrounding the first edge outline, each second microflake is identifiedby a second binary sequence encoded by a second edge outline on theplane, and bits in the second binary sequence are encoded at respectivepredefined locations surrounding the second edge outline, each of thefirst edge outline and the second edge outline includes a first-typenotch which indicates a starting point and a direction along which thefirst binary sequence or the second binary sequence is to be read, andeach of the first microflake and the second microflake is furtheridentified by a group identifier which is encoded by adjacent angles ofthe first-type notch, and wherein different combinations of the adjacentangles encode different group identifiers.
 8. The apparatus of claim 7,wherein each microflake of the first microflakes and the secondmicroflakes includes a substantially transparent polymer layer.
 9. Theapparatus of claim 7, wherein each microflake of the first microflakesand the second microflakes is magnetic.
 10. The apparatus of claim 7,wherein each of the first edge outline and the second edge outlineincludes the first-type notch as an orientation indicator, and whereinat least one of the first edge outline and the second edge outlineincludes a second-type notch representing a binary value in the firstbinary sequence or the second binary sequence.
 11. The apparatus ofclaim 8, wherein a magnetic metal strip is embedded within and locatedat a geometric center of the substantially transparent polymer layer.12. The apparatus of claim 7, wherein presence of a second-type notch ata predefined location of each of the first edge outline and the secondedge outline corresponds to a first binary value, and absence of thesecond-type notch at the predefined location of each of the first edgeoutline and the second edge outline corresponds to a second binaryvalue.